The actin cytoskeleton is important in the machinery of motile and non- motile cells. Different cytoskeletal assemblies fulfill different roles, and their different functions are reflected in their different designs. Cytoskeletal design is mediated by the proportion, kind and organization of the actin-binding proteins present. We have been studying the organization and assembly of the cytoskeletons of specialized cells because their cytoskeletons are relatively simple in composition and structure. Several recent advances have opened a pathway to obtaining detailed models for in vivo cytoskeletal structures, in particular, actin bundles. The discovery that many of the proteins are modular in design permits us to dissect the cytoskeleton into parts. We propose in particular to study the fimbrin and fimbrin/villin bundles found respectively in the specialized hair cells of the inner ear and in the brush border cells of the intestine. We propose to produce 3-D maps of act in complexed to the cloned act in- binding modules found in fimbrin, villin, or their homologues. These maps will give us the fimbrin- or villin-binding sites on actin, the conformation of act in, and a map of the module itself. Next we will obtain maps of 2-D arrays of F-actin crosslinked by intact fimbrin or villin or both. We propose then to dock the maps of the module-decorated filaments into the maps obtained from the 2-D arrays and thereby derive a 3-D model for a row of crossbridged filaments. This model in turn will be used to construct a 3-D model of an in vivo 3-D bundle. As atomic structures become available for crossbridges, we will be able to dock these into our maps. In this way we can begin the long task of constructing atomic models for the cytoskeleton. No one technique nor any one lab can accomplish all of this, but as evidenced in the recent atomic model for the acto-S1 complex, it can be done by a combination of techniques and labs. We also propose to explore two other actin cytoskeletons. One is the dynamic actin gel found in cells infected by pathogenic bacteria. These gels are essential to the spread of infection. The second is a highly ordered actin bundle found in the bristles of the fruit fly. This is an exciting system because of the availability of mutants and the ability to carry out genetic analysis.